1 Whole genome analysis of a schistosomiasis-transmitting freshwater snail
2 Adema, Coen M.; Hillier LaDeana W.; Jones, Catherine S.; Loker, Eric S.; Knight, Matty; Minx,
3 Patrick; Oliveira, Guilherme; Raghavan, Nithya; Shedlock, Andrew; Amaral, Laurence Rodrigues
4 do; Arican-Goktas, Halime D.; Assis, Juliana; Baba, Elio Hideo; Baron, Olga Lucia; Bayne,
5 Christopher J.; Bickham-Wright, Utibe; Biggar, Kyle K.; Blouin, Michael; Bonning, Bryony C.;
6 Botka, Chris; Bridger, Joanna M.; Buckley, Katherine M.; Buddenborg, Sarah K.; Caldeira, Roberta
7 Lima; Carleton, Julia; Carvalho, Omar S.; Castillo, Maria G.; Chalmers, Iain W.; Christensens,
8 Mikkel; Clifton, Sandy; Cosseau, Celine; Coustau, Christine; Cripps, Richard M.; Cuesta-Astroz,
9 Yesid; Cummins, Scott; di Stephano, Leon; Dinguirard, Nathalie; Duval, David; Emrich, Scott;
10 Feschotte, Cédric; Feyereisen, Rene; FitzGerald, Peter; Fronick, Catrina; Fulton, Lucinda; Galinier,
11 Richard; Geusz, Michael; Geyer, Kathrin K.; Giraldo-Calderon, Gloria; Gomes, Matheus de Souza;
12 Gordy, Michelle A.; Gourbal, Benjamin; Grossi, Sandra; Grunau, Christoph; Hanington, Patrick C.;
13 Hoffmann, Karl F.; Hughes, Daniel; Humphries, Judith; Izinara, Rosse; Jackson, Daniel J.; Jannotti-
14 Passos, Liana K.; Jeremias, Wander de Jesus; Jobling, Susan; Kamel, Bishoy; Kapusta, Aurélie;
15 Kaur, Satwant; Koene, Joris M.; Kohn, Andrea B; Lawson, Dan; Lawton, Scott P.; Liang, Di;
16 Limpanont, Yanin; Lockyer, Anne E.; Lovato, TyAnna L.; Liu, Sijun; Magrini, Vince; McManus,
17 Donald P.; Medina, Monica; Misra, Milind; Mitta, Guillaume; Mkoji, Gerald M.; Montague,
18 Michael J.; Montelongo, Cesar; Moroz, Leonid L; Munoz-Torres, Monica C.; Niazi, Umar; Noble,
19 Leslie R.; Pais, Fabiano; Papenfuss, Anthony T.; Peace, Rob; Pena, Janeth J: Pila, Emmanuel A.;
20 Quelais, T; Raney, Brian J.; Rast, Jonathan P.; Ribeiro, Fernanda; Rollinson, David; Rotgans,
21 Bronwyn; Routledge, Edwin J.; Ryan, Kathryn M.; Scholte, Larissa; Silva, Francislon; Storey,
22 Kenneth B; Swain, Martin; Tennessen, Jacob A.; Tomlinson, Chad; Trujillo, Damian L.; Volpi,
23 Emanuela V.; Walker, Anthony J.; Wang, Tianfang; Wannaporn, Ittiprasert; Warren, Wesley C.; 24 Wu, Xiao-Jun; Yoshino, Timothy P.; Yusuf, Mohammed; Zhang, Si-Ming; Zhao, Min; Wilson,
25 Richard K.
26 27 Summary
28 Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni.
29 With the World Health Organization's goal to eliminate schistosomiasis as a global health problem
30 by 2025, there is now renewed emphasis on snail control. Our characterization of the genome of
31 Biomphalaria glabrata, a lophotrochozoan protostome, provides timely and important information
32 on snail biology. We describe phero-perception, stress resistance, immune function and multi-level
33 regulation of gene expression that support the persistence of B. glabrata in aquatic habitats and
34 define this species as a suitable snail host for S. mansoni. We identify several potential targets for
35 developing novel control measures aimed at reducing snail-mediated transmission of
36 schistosomiasis.
37 38 The fresh water snail Biomphalaria glabrata (Lophotrochozoa, Mollusca) is of medical relevance as
39 this Neotropical gastropod contributes as obligate intermediate host of Schistosoma mansoni
40 (Lophotrochozoa, Platyhelminthes) to transmission of the neglected tropical disease (NTD) human
41 intestinal schistosomiasis1. An S. mansoni miracidium initiates a chronic parasite-snail infection that
42 alters B. glabrata immunity and metabolism, causing parasitic castration such that the snail does not
43 reproduce but instead supports generation of cercariae, the human-infective stage of S. mansoni.
44 Patently infected snails continuously release free-swimming cercariae that penetrate the skin of
45 humans that they encounter in their aquatic environment. Inside the human definitive host, S.
46 mansoni matures to adult worms that reproduce sexually in the venous system surrounding the
47 intestines, releasing eggs, many of which pass through the intestinal wall and are deposited in water
48 with the feces. Miracidia hatch from the eggs and complete the life cycle by infecting another B.
49 glabrata. Related Biomphalaria species transmit S. mansoni in Africa. Schistosomiasis is chronically
50 debilitating; estimates of disease burden indicate that disability-adjusted life years (DALYs) lost due
51 to morbidity rank schistosomiasis second only to malaria among parasitic diseases in impact on
52 global human health2.
53 In the absence of a vaccine, current control measures emphasize mass drug administration (MDA) of
54 praziquantel (PZQ), the only drug available for large-scale treatment of schistosomiasis3.
55 Schistosomes, however, may develop resistance and reduce the effectiveness of PZQ4. Importantly,
56 PZQ treatment does not protect humans against rapid re-infection following exposure to water-borne
57 cercariae released from infected snails. Snail-mediated parasite transmission must be interrupted to
58 achieve long-term sustainable control of human schistosomiasis5. The World Health Organization
59 has set a strategy for the global elimination of schistosomiasis as a public health threat by the year
60 2025 that recognizes both MDA approaches and targeting of the snail intermediate host as crucial 61 toward achieving this goal6. This significant undertaking provides added impetus for detailed study
62 of B. glabrata.
63 Genome analyses of B. glabrata also provide evolutionary insights by increasing the relatively few
64 taxa of the Lophotrochozoa that have been characterized. To date a majority of lophotrochozoan
65 genomes have been derived from parasitic, sessile, or marine animals (i.e. platyhelminths, leech,
66 bivalve, cephalopod, polychaete)7-11. Thus, B. glabrata likely possesses novel genomic features that
67 characterize a free-living lophotrochozoan from a freshwater environment. Here we characterize the
68 B. glabrata genome and describe biological properties that enable the snail’s persistence in aquatic
69 environments and define this snail as a suitable host for S. mansoni, including aspects of immunity
70 and gene regulation. These efforts, we anticipate, will foster developments to interrupt snail-
71 mediated parasite transmission in support of schistosomiasis elimination.
72 The B. glabrata genome comprises eighteen chromosomes (Supplementary Text 1 and
73 Supplementary Fig. 1-3) and has an estimated size of 916Mb12. We assembled the genome of BB02
74 strain B. glabrata13 from short reads, Sanger sequences, and 454 sequence data (~27.5X coverage)
75 and used a linkage map to assign genomic contigs to linkage groups (Supplementary Text 2 and
76 Supplementary Table 1). We captured transcriptomes from twelve different tissues (Illumina
77 RNAseq) and mapped these reads to the assembly to aid annotation (Supplementary Text 3,
78 Supplementary Figs. 4-8 and Supplementary Tables 2-7).
79 Olfaction in an aquatic environment
80 Aquatic molluscs employ proteins for communication; e.g. Aplysia attracts conspecifics using water-
81 soluble peptide pheromones14. We collected B. glabrata proteins from snail conditioned water
82 (SCW) and following electrostimulation (ES), which induces rapid release of proteins. NanoHPLC-
83 MS/MS identified 177 secreted proteins shared among 533 proteins from SCW and 232 proteins 84 from ES (Supplementary Table 8). Detection of an ortholog of temptin, which plays a key role in
85 pheromone attraction of Aplysia15, suggests an operational pheromone sensory system in B.
86 glabrata. To gain insight into the mechanisms for pheromone perception, we analyzed the B.
87 glabrata genome for olfactory G-protein coupled receptor (GPCR)-like supergene families and
88 identified 242 seven transmembrane domain GPCR-like genes belonging to fourteen subfamilies that
89 are clustered in the genome (Supplementary Text 4, Supplementary Figs. 9,10 and Supplementary
90 Table 9). RT-PCR and in situ hybridization confirmed expression of a GPCR-like gene within the
91 mollusc’s tentacles, known to be involved in chemosensation (Figure 1). This communication
92 system allows B. glabrata to detect conspecifics in aquatic environments but may have a tradeoff
93 effect by potentially exposing the snail as a suitable target for pathogens. 94
95 Fig 1. Candidate chemosensory receptors of B. glabrata. (A) Scaffold 39 contains fourteen 96 candidate chemosensory receptor genes (BgCRa-n). Most encode 7-transmembrane domain proteins, 97 BgCRm and BgCRn are truncated to six-transmembrane domains. Genes occur in both directions. 98 (B) Phylogenetic analysis of scaffold 39 chemosensory receptors. (C) Schematic of receptor showing 99 conserved and invariable amino acids, transmembrane domains I-VII; and location of glycosylation 100 sites. (D) Scanning electron micrograph showing anterior tentacle, with cilia covering the surface. 101 (B) RT-PCR gel showing amplicon for BgCR509a and actin from B. glabrata tentacle. (F, G) In situ 102 hybridization showing sense (negative control) and antisense localization of BgCR509a in anterior 103 tentacle section (purple). 104 105 Stress responses and immunity
106 Biomphalaria glabrata endures environmental insults, including biotic and abiotic stress from heat,
107 cold, xenobiotics, pollutants, injury and infection. Additional to previous reports of Capsaspora16 as
108 single-cell eukaryote endosymbiont, we noted from the sequenced material an unclassified
109 mycoplasma or related mollicute bacteria and viruses (Supplementary Text 5,6, Supplementary
110 Figs. 11,12 and Supplementary Table 10). For anti-stress responses, B. glabrata genome has five
111 families of heat shock proteins (HSP), including Hsp20, Hsp40, Hsp60, and Hsp 90. The Hsp70 gene
112 family is the largest with six multi-exon genes, five single exon genes, and over ten pseudogenes
113 (Supplementary Text 7, Supplementary Figs. 13-16, Supplementary Table 11). These genes are
114 retained in B. glabrata embryonic (Bge) cells, the only available molluscan cell line17, such that anti-
115 stress function in B. glabrata can be investigated in vitro using cell cultures (Supplementary Text 8,
116 Supplementary Figs. 17-21, and Supplementary Table 12,13).
117 The B. glabrata genome contains about 99 genes encoding heme-thiolate enzymes (CYP
118 superfamily) for detoxifying xenobiotics. All major animal cytochrome P450 clans are represented,
119 including ~33 CYP2 genes, ~35 CYP3 genes, and ~7 CYP4 genes. The presence of 18 genes of the
120 mitochondrial clan suggests that molluscs, like arthropods, but unlike vertebrates, utilize
121 mitochondrial P450s for detoxification18. Tissue-specific expression (e.g. four uniquely in ovotestis)
122 suggests that fifteen P450 genes serve specific biological processes. Cataloguing the CYPome sets
123 the groundwork for rational design of selective molluscicides, e.g. by inhibiting unique P450s or
124 using B. glabrata-specific P450s for bioactivation of pro-molluscicides (Supplementary Text 9).
125 Biomphalaria glabrata possesses a diversity of pattern recognition receptors (PRRs) for immune
126 recognition of pathogens19. This includes 56 Toll-like receptor (TLR) genes, of which 27 encode
127 complete TLRs (Fig 2, Supplementary Text 10 and Supplementary Table 14), associated with a TLR 128 signaling network for transcriptional regulation through NF-B transcription factors (Supplementary
129 Text 11, Supplementary Fig. 22 and Supplementary Table 16). Like other lophotrochozoans, B.
130 glabrata shows a moderate expansion of TLR genes relative to mammals and insects which have
131 ~10 TLRs20. Other PRRs include eight peptidoglycan recognition-binding proteins (PGRPs), a single
132 Gram-negative binding protein (GNBP) and seventeen genes encoding for single scavenger receptor
133 cysteine-rich (SRCR) domains (Supplementary Table 15). A prominent category of B. glabrata
134 PRRs consists of fibrinogen-related proteins (FREPs), plasma lectins that are somatically mutated to
135 yield uniquely diversified FREP repertoires in individual snails21. The B. glabrata germline includes
136 four FREP genes comprising one immunoglobulin (IgSF) domain and one C-terminal fibrinogen
137 (FBG)-like domain, including one gene with an N-terminal PAN_AP domain, and twenty FREP
138 genes with two upstream IgSF domains preceding an FBG domain. FREP genes cluster in the
139 genome, often accompanied by partial FREP-like sequences (Supplementary Text 12 and
140 Supplementary Figs. 23-26). A proteomics level study of anti-parasite immunity showed that some
141 FREPs exhibiting binding reactivity to S. mansoni derive from different gene families between
142 susceptible and resistant B. glabrata strains. Moreover, a FREP-like lectin that contains a galectin
143 instead of a C-terminal fibrinogen domain (designated galectin-related protein; GREP22) is uniquely
144 associated with the resistant snail phenotype (Supplementary Text 8).
145 We identified several cytokines, including twelve homologs of IL-17, four MIF homologs, and
146 eleven TNF molecules (Supplementary Text 10, Supplementary Table 15). Orthologs of complement
147 factors indicate that B. glabrata can mount complement-like responses to pathogens (Supplementary
148 Text 13, Supplementary Figs. 27,28 and Supplementary Tables 17-28). We discovered an 149 150 Fig 2. TLR genes in B. glabrata. (A) Analysis of the (only complete) TIR domains from BgTLRs 151 identified seven classes (Neighbor-joining tree shown). Bootstrap values shown for 1,000 replicates. 152 Comparisons included TLRs from A. californica (Ac), L. gigantea (Lg), Mytilus galloprovincialis 153 (Mg), and C. gigas (Cg). The presence or absence of orthologs of each class in each molluscan 154 species is indicated. A representative of the B. glabrata class 1/2/3 clade is present within A. 155 californica, but is independent of the B. glabrata TLR classes (indicated by the large pink box). 156 Grey font indicates pseudogenes or partial genes. (B) B. glabrata has both single cysteine cluster 157 (scc; blue line)- and multiple cysteine cluster (mcc; orange line) TLRs. Domain structures are 158 shown for each of BgTLR class. BgTLRs consist of an LRRNT (orange hexagon), a series of LRRs 159 (ovals), a variable region (curvy line), LRRCT (yellow box), and transmembrane domain, and an 160 intracellular TIR domain (red hexagon). The dark blue ovals indicate well defined LRRs (predicted 161 by LRRfinder23); light blue ovals are less confident predictions. Each of the two class 7 BgTLRs has 162 a distinct ectodomain structure. The numbers of complete, pseudogenes () and partial and genes 163 are indicated for each class. 164 165 extensive toolkit for apoptosis, a response that can regulate invertebrate immune defense24. This
166 includes at least 50 genes encoding for Baculovirus IAP Repeat (BIR) domain-containing caspase
167 inhibitors, localized among 26 genomic scaffolds. The expansion of this gene family in molluscs (17
168 genes in Lottia gigantean, 48 in Crassostrea gigas), relative to other animal clades, suggests
169 important regulatory roles in apoptosis and innate immune responses25 (Supplementary text 14,
170 Supplementary Figs. 29-31 and Supplementary Table 29). Finally, B. glabrata possesses a gene
171 complement to metabolize reactive oxygen species (ROS) and nitric oxide (NO), that are generated
172 by hemocytes to exert cell-mediated cytotoxicity toward pathogens, including schistosomes
173 (Supplementary Text 15, Supplementary Fig. 32 and Supplementary Table 30).
174 Searches for antimicrobial peptide (AMP) sequences of B. glabrata indicated only a single macin-
175 type gene family, comprising six biomphamacin genes. While the AMP arsenal is reduced compared
176 to other invertebrate species (e.g., bivalve molluscs have multiple AMP gene families26), B. glabrata
177 possesses several multigenic families of antibacterial proteins including two achacins, five
178 LBP/BPIs, and 21 biomphalysins (Supplementary Text 16,17, Supplementary Figs. 33,34 and
179 Supplementary Tables 31,32). Gaps in functional annotation limit our interpretation of immune
180 function in B. glabrata (Supplementary Text 18 and Supplementary Table 33,34). Computational
181 analysis indicated that 15% of the predicted proteome consists of (583) secreted proteins. Immune-
182 relevant tissues showed high expression of 100 secreted proteins, including FREP3, other lectins and
183 novel proteins that potentially represent candidate immune factors for future study. (Supplementary
184 text 3, Supplementary Fig. 7, Supplementary Tables 5-6).
185 Regulation of biological processes
186 Epigenetic regulation allows dynamic use of genes contained in the B. glabrata genome27-29. The
187 chromatin-modifying enzymes include class I and II histone methyltransferases, LSD-class and 188 Jumonji-class histone demethylases, class I – IV histone deacetylases, and GNAT, Myst and CBP
189 superfamilies of histone acetyltransferases. We identified homologs of DNA (cytosine-5)-
190 methyltransferases 1 and 2 (no homolog of DNMT3), as well as putative methyl-CpG binding
191 domain proteins 2/3. In silico analyses predicted a mosaic type of DNA methylation, as is typical for
192 invertebrates (Supplementary Text 19, Supplementary Figs. 35-39, Supplementary Table 35). The
193 potential role of DNA methylation in B. glabrata reproduction and S. mansoni interactions is
194 reported elsewhere (K.G.G., U.H.N., D.D., Ce.C., C.T., I.W.C., M.T.S., U.B.-W., Sabrina E.
195 Munshi, C.G., T.P.Y. and K.F.H., in preparation).
196 The B. glabrata genome also provides the protein machinery for biogenesis of microRNA (miRNAs)
197 to regulate gene expression. Moreover, two computational methods independently predicted the
198 same 95 pre-miRNAs, encoding 102 mature miRNAs. Of these, 36 predicted miRNAs were
199 observed within our transcriptome data, while 53 displayed ≥ 90% nucleotide identity (with 25 at
200 100%) to L. gigantea miRNAs. One bioinformatics pipeline predicted 107 additional pre-miRNAs
201 unique to B. glabrata. Several B. glabrata miRNAs are predicted to regulate transcripts for
202 processes unique to snail biology, including secretory mucosal proteins and shell formation that may
203 present possible targets for control of B. glabrata (Supplementary Text 20,21, Supplementary Figs.
204 40-67, Supplementary Tables 36-45).
205 Aspects of B. glabrata biology are periodic30, likely due to control of circadian timing mechanisms.
206 We identified seven candidate clock genes in silico, including a gene with strong similarity to the
207 period gene of A. californica. Modification of expression of these putative clock genes may interrupt
208 circadian rhythms and affect feeding and egg-laying of B. glabrata (Supplementary Text 22).
209 Neuropeptides expressed within the nervous system coordinate the complex physiology of B.
210 glabrata, including regulation of male and female functions in this simultaneous hermaphrodite 211 snail. In silico searches identified 43 neuropeptide precursors in B. glabrata that were predicted to
212 yield over 250 mature signaling products. Neuropeptide transcripts occurred in multiple tissues, yet
213 some were most prominent within terminal genitalia (49%) and the CNS (56%), or even specific to
214 the CNS, including gonadotropin-releasing hormone (GnRH) and insulin-like peptides 2 and 3
215 (Supplementary Text 23, Supplementary Fig. 68, Supplementary Tables 46-48).
216 A role of steroid hormones in reproduction of hermaphrodite snails with male and female
217 reproductive organs remains speculative. Biomphalaria glabrata has a CYP51 gene to biosynthesize
218 sterols de novo, yet we found no orthologs of genes involved in either vertebrate steroid or arthropod
219 ecdysteroid biosynthesis. The lack of CYP11A1 suggests that B. glabrata cannot process cholesterol
220 to make vertebrate-like steroids. The absence of aromatase (CYP19), required for the formation of
221 estrogens, is particularly enigmatic as molluscs possess homologs of mammalian estrogen receptors.
222 Characterization of snail-specific aspects of steroidogenesis may identify targets to disrupt
223 reproduction toward control of snails. (Supplementary Text 24, Supplementary Fig. 69,
224 Supplementary Table 49).
225 The reproductive physiology of hermaphroditic snails is also modulated by male accessory gland
226 proteins (MAGPs), which are delivered with spermatozoa to augment fertilization success31. The B.
227 glabrata genome has sequences matching one such protein, Ovipostatin (LyAcp10), but none of the
228 other MAGPs identified in Lymnaea stagnalis32. Perhaps MAGPs evolved rapidly and are putatively
229 taxon-specific (Supplementary Text 25, Supplementary Fig. 70 and Supplementary Table 50).
230 KEGG Enrichment analyses33 identified metabolic and biochemical pathways in B. glabrata and
231 revealed distinct organ specific patterns of gene expression (Supplementary Text 3,26, Figs. 8, 71-
232 80, Supplementary Tables 3,51). Half of 5,000 annotated sequences were assigned to 200
233 biochemical pathways (Supplementary Text 3 and Supplementary Tables 2,3). Mapping biological 234 processes onto the snail anatomy helps interpreting B. glabrata’s responses to environmental insults
235 and pathogens.
236 For survival in B. glabrata, S. mansoni alters the snail host physiology, possibly by interfering with
237 the extracellular signal-regulated kinase (ERK) pathway34 and other signaling pathways. Eukaryotic
238 protein kinases (ePKs) mediate signal transduction through phosphorylation in complex networks,
239 and protein phosphatases counteract these effects towards effective signaling. Similarity searches for
240 conserved domains identified 240 B. glabrata ePKs encompassing all main types of animal ePKs
241 (Supplementary Text 3 and Supplementary Fig. 6). We also identified 60 putative protein
242 phosphatases comprising ~36 protein Tyr phosphatases (PTPs) and ~24 protein Ser/Thr
243 phosphatases (PSPs) (Supplementary Text 27, Supplementary Figs. 81-83). These sequences can be
244 studied for understanding control of homeostasis, particularly in the face of environmental and
245 pathogenic insults encountered by B. glabrata (Figure 3).
246 247
248 Fig 3. MAPK signaling pathway in B. glabrata. Blue shaded blocks correspond to proteins identified 249 in the B. glabrata predicted proteome, white blocks represent mammalian proteins without homologs 250 in the snail proteome. The +p signal represents phosphorylation and the –p signal represents protein 251 dephosphorylation. 252 253 Bilaterian evolution
254 We searched the B. glabrata genome for core cardiac-specification and -differentiation genes. A
255 previously characterized short cDNA sequence from snail heart RNA led to identification of
256 BGLB012592 as the Biomphalaria ortholog of tin/Nkx2.5 35. Similarity searches with Drosophila
257 orthologs identified most of the core cardiac regulatory factors and structural genes in the B.
258 glabrata genome (Supplementary Text 28, Supplementary Table 52), with enriched expression of
259 these genes in cardiac tissues (Figure 4). These results from a lophotrochozoan, in conjunction with
260 ecdysozoans and deuterostomes, further support the hypothesis that a primitive heart-like structure,
261 which developed through the actions of a core heart toolkit, was present in the urbilaterian ancestor.
262 Actins are conserved proteins that function in cell motility (cytoplasmic actins) and muscle
263 contraction (sarcomeric actins)36. The clustering across seven scaffolds suggests that some of the ten
264 B. glabrata actin genes arose through tandem duplication. Expression across all tissues indicates that
265 four genes encode cytoplasmic actins (Figure 4). Protein sequence comparisons placed all B.
266 glabrata actins as most closely related to mammalian cytoplasmic rather than sarcomeric actins
267 (Supplementary Text 29 and Supplementary Table 53), a pattern also observed for all six actin genes
268 of D. melanogaster 37. The actin genes of B. glabrata and other molluscs were most similar to
269 paralogs within their own genomes, rather than to other animal orthologs (Figure 4). One
270 interpretation is that actin genes diverged independently multiple times in molluscs, similar to an
271 earlier hypothesis for independent actin diversification in arthropods and chordates38. Alternatively,
272 a stronger appearance of monophyly than really exists may result if selective pressures due to
273 functional constraints keep actin sequences similar within a genome, for example if the encoded
274 proteins have overlapping functions. 275 A D 276 277 278 279 280 281 282 283 284 285 286 B 287 288 289 290 291 292 293 294 295 296 297 C 298 299 300 301 302 303 304 305 306 307 Fig 4. Expression of cardiac genes and actin genes in B. glabrata tissues determined through RNA- 308 seq analysis. (A) cardiac regulatory genes. (B) cardiac structural genes. (C) Relative expression of 309 actin genes in B. glabrata tissues. (D) Phylogenetic relationships of actin genes. 310 311 AG - Albumen gland; BUC - buccal mass; CNS - central nervous system; DG - digestive gland; FOOT – headfoot; HAPO - 312 heart/APO; KID – kidney; MAN - mantle edge; OVO – ovotestes; SAL - salivary glands; STO - stomach; TRG - terminal genitalia 313 Amphimedon – marine sponge; Haliotis – abalone; Ciona – sea squirt; Hirudo – leech; Crassostrea – oyster; Biomphalaria – pond 314 snail; Homo – human; Drosophila – fruit fly. 315 316 We analyzed the transcriptomic data for B. glabrata genes involved in biomineralization. Of 1,211
317 transcripts that were >2-fold up-regulated in the mantle relative to other tissues, 34 shared similarity
318 with molluscan sequences known to be involved in shell formation and biomineralization. Another
319 177 candidate sequences putatively involved in shell formation including eighteen genes (10.2%)
320 with similarity to sequences of shell forming secretomes of other marine and terrestrial molluscs
321 were identified from the entire mantle transcriptome (Figure 5). Like previous studies, our results
322 show both diverse and conserved aspects of molluscan shell-forming strategies. Highly conserved
323 components of the molluscan shell forming toolkit include carbonic anhydrases and tyrosinases9.
324 (Supplementary Text 30, Supplementary Fig. 84, Supplementary Tables 54). 325
326 Fig 5. Circos diagram of 177 mantle specific, secreted Biomphalaria gene products compared 327 against six other molluscan shell forming proteomes (BLASTp threshold ≤10e-6). Protein pairs that 328 share sequence similarity in the top quartile are linked in green, the second quartile is linked in blue, 329 third quartile has orange links and lowest quartile of similarity has red links. Marine species are 330 circled in blue, freshwater species in green, terrestrial species in red. Percentages (proportions in 331 brackets) indicate the number of proteins that shared similarity with a Biomphalaria shell forming 332 candidate gene. 333 334 Repetitive landscape
335 Repeat content analysis showed that 44.8% of the B. glabrata assembly consists of transposable
336 elements (TEs; Fig 6, Supplementary Text 31, Supplementary Figs. 87-87 and Supplementary Table
337 55), higher than observed from other molluscs: Pacific oyster, C. gigas (36%)9, owl limpet, L.
338 gigantea (21%)8, sea hare, A. californica (30%)39, and comparable to Octopus bimaculoides (43%)11.
339 The fraction of unclassified elements in B. glabrata was high (17.6%). Most abundant classified
340 repeats were LINEs, including Nimbus40 (27% of TEs, 12.1% of the genome), and DNA TEs (17.7%
341 of TEs, 8% of the genome), long terminal repeats (LTRs) were a small percentage of the repetitive
342 elements (6% of TEs, 1.7% of the genome). Non-mobile simple repeats comprised 2.6% of the
343 genome with an abundance of short dinucleotide satellite motifs. Divergence analyses of element
344 copy and consensus sequences indicated that DNA TEs were not recent invaders of the B. glabrata
345 genome, no intact transposases were detected. A hAT DNA transposon of B. glabrata (~1000
346 copies) has significant identity with SPACE INVADERS (SPIN) which horizontally infiltrated a
347 range of animal species, possibly through host-parasite interactions41. Overall, our results reinforce a
348 model in which diverse repeats comprise a large fraction of molluscan genomes.
349 350
351
352 Fig 6. Transposable element (TE) landscape of B. glabrata. (A) Left: proportion of DNA annotated 353 as TE (in black) or not annotated (white). Right: TE composition by class. Numbers represent the 354 percentage of the genome corresponding to each class. (B) Evolutionary view of TE landscape. For 355 each class, cumulative amounts of DNA (in Mb) are shown in function of the percentage of 356 divergence to the consensus (by bins of 1%, first one being ≥0 and <1; see Methods). Percentage of 357 divergence to the consensus is used as a proxy for age: the older the invasion of the TE is, the more 358 copies will have accumulated mutations (higher percentage of divergence, right of the graph). 359 Conversely, sequences corresponding to youngest elements show little divergence to consensus (left 360 of the graph). RC: rolling circle. 361 362 Concluding remarks
363 The genome of the Neotropical freshwater snail B. glabrata expands insights into animal biology by
364 further defining the lineage of the Lophotrochozoa relative to Ecdysozoa and Deuterostomia among
365 higher animals. An important rationale for analysis of the genome of B. glabrata pertains to its role
366 in transmission of S. mansoni in the New World. Moreover, most of the world’s cases of S. mansoni
367 infection occur in sub-Saharan Africa where other Biomphalaria species are responsible for
368 transmission, most notably Biomphalaria pfeifferi. Due to a shared common ancestor, B. glabrata
369 likely provides a good representation of the genomes of African Biomphalaria species42,43 . This
370 notion is supported by at least 90% sequence identity shared among 196 assembled transcripts
371 collected from B. pfeifferi (Illumina RNAseq) with the transcriptome of B. glabrata (Supplementary
372 Text 32 and Supplementary Tables 56-58). This report provides novel details on the biological
373 properties of B. glabrata and points to potential strategies for more effective surveillance and control
374 efforts against Biomphalaria to limit the transmission of schistosomiasis. 375 Methods
376 The genetic material used for sequencing the hermaphroditic freshwater snail B. glabrata was
377 derived from multiple snails of the BB02 strain, established at the University of New Mexico, USA
378 from a filed isolate collected from Minas Gerais, Brazil. Using a genome size estimate of 0.9-1Gb12,
379 we sequenced fragments (15X coverage), 3kb long inserts (10X), and 8kb long inserts (3X) with
380 reads generated on Roche 454 instrumentation, plus 1X coverage from plasmids and 0.1X from
381 bacterial artificial chromosome (BAC) ends13 on the ABI3730xl. Reads were assembled using
382 Newbler (v2.6)44. Additional sequencing reads were collected using Illumina instrumentation,
383 including 200bp short inserts (45X), 3kb long inserts (15X), and 8kb long inserts (10X), and
384 assembled de novo using SOAP de novo (v1.0.5)45. Finally, the Newbler assembly was merged with
385 the SOAP assembly using GAA46.
386 Redundant contigs in the merged assembly were collapsed and gaps between contigs were closed
387 through iterative rounds of Illumina mate-pair read alignment and extension using custom scripts.
388 We removed from the assembly all contaminating sequences, trimmed vectors (X), and ambiguous
389 bases (N). Shorter contigs (≤200bp) were removed prior to public release.
390 In the creation of the linkage group AGP files, we identified all scaffolds (145Mb total) that were
391 uniquely placed on a single linkage group (Supplementary Text 2 and Supplementary Table 1).
392 Because of low marker density and scaffolds could not be ordered and oriented within linkage
393 groups. The final draft assembly, (NCBI: ASM45736v1) is comprised of 331,400 scaffolds with an
394 N50 scaffold length of 48kb and an N50 contig length of 7.3kb. The assembled coverage (Newbler)
395 is 27.5X, and the assembly spans over 916Mb (with a coverage of 98%, 899Mb of sequence with
396 ~17Mb of estimated gaps). The draft genome sequence of Biomphalaria glabrata was aligned with
397 assemblies of Lottia and Aplysia (http://genome-test.cse.ucsc.edu/cgi- 398 bin/hgGateway?hgsid=389472876_vEhDXpybetKHBbwuzZFov0KE6Qhl&clade=other&org=Snail
399 &db=0) and also deposited in the DDBJ/EMBL/GenBank database (Accession Number
400 APKA00000000.1). It includes the genomes of an unclassified mollicute and viruses
401 (Supplementary 5 and 6: Accession Numbers CP013128, KT728710-12). Total RNA was extracted
402 from 12 different tissues dissected from multiple adult BB02 snails. Illumina RNAseq was used to
403 generate tissue-specific transcriptomes for albumen gland (AG); buccal mass (BUC); central nervous
404 system (CNS); digestive gland/hepatopancreas (DG/HP); muscular part of the headfoot (FOOT);
405 heart including amebocyte producing organ (HAPO); kidney (KID); mantle edge (MAN); ovotestis
406 (OVO); salivary gland (SAL); stomach (STO); terminal genitalia (TRG). The genome assembly was
407 also deposited in Vectorbase47, (https://www.vectorbase.org/organisms/biomphalaria-glabrata).
408 Computational annotation using Maker248 yielded 14,423 predicted gene models, including 96.5% of
409 the 458 sequences from the CEGMA core set of eukaryotic genes49. RNAseq data were mapped to
410 the genome assembly and Web Apollo50 was applied to aid manual annotation of genes of interest by
411 contributors. Methods and results are described in the Supplementary Information. 412 References
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526 Acknowledgments
527 We thank S. Newfeld for discussion of actin evolution. Sequence characterization of the
528 Biomphalaria glabrata genome was funded by National Institutes of Health (NIH) grant HG003079
529 to R.K.W. and McDonnell Genome Institute, Washington University School of Medicine. Further
530 work was supported by FAPEMIG (RED-00014-14) and CNPq (304138/2014-2, 309312/2012-4) to
531 G.O.. K.F.H. and M.S. acknowledge the support of the UK BBSRC (BB/K005448/1) K.F.H. and
532 M.S. acknowledge the support of BBSRC (BB/K005448/1) and CNPq (503275/2011-5) to R.L.C.
533 C.B. acknowledges the support of NIH grant AI016137. J.M.K. acknowledges support from the
534 Research Council for Earth and Life Sciences (ALW; 819.01.007) with financial aid from the
535 Netherlands Organization for Scientific Research (NWO). S.E. acknowledges NIAID contract 536 HHSN272201400029C. C.M.A. was supported by NIH grant P30GM110907from the National
537 Institute of General Medical Sciences (NIGMS). R.M.C. acknowledges NIH grant GM061738 and
538 support from the American Heart Association, Southwest Affiliate (14GRNT20490250). D.T. was
539 supported by NIH grant R25 GM075149. M.K. acknowledges NIH-NIAID grant R01-AI063480.
540 C.F. acknowledges NIH grant R01-GM077582. D.J.J. acknowledges D.F.G grant JA2108/1-2.
541 E.S.L. acknowledges NIH grant P30GM110907 and ROI AI101438. BG acknowledges ANR JCJC
542 INVIMORY (ANR-13-JSV7-0009). K.M.B and J.P.R acknowledge Natural Sciences and
543 Engineering Research Council (NSERC 312221) and the Canadian Health Institutes for Research
544 (CIHR MOP74667). C.S.J, L.R.N, S.J, E.J.R, S.K and A.E.L acknowledge funding from NC3R’s
545 Grant (ref GO900802/1).
546
547 Author Contributions
548 C.M.A., E.S.L., M.K., N.R. conceived the study, scientific objectives. C.M.A. led the project and
549 manuscript preparation with input from steering committee members M.K., C.S.J., G.O., P.M.,
550 L.W.H., A.S., E.S.L., assisted by S.E. O.C. provided field collected snails. C.M.A. and E.S.L
551 cultured snails and provided materials. P.M., L.W.H, S.C., L.F., W.C.W, R.K.W., V.M., C.T
552 developed the sequencing strategy, managed the project, conducted assembly and evaluation. B.R.
553 performed genome alignments. M.C., D.H., S.E., G.G-C. and D.L. perfomed the genebuild, managed
554 metadata, performed genome annotation and data analysis, and facilitated Community Annotation
555 with M.C.M-T. H.D. A-G., M.Y., E.V.V., M.K. and J.M.B. performed Karyotyping and FISH
556 analysis. J.A.T and M.B. performed linkage mapping. G.O., F.P., F.R., J.A., I.R., Y.C., S.G., F.S.
557 and L.S. performed computational analyses of genomic, proteomic, and transcriptomic data, SNP
558 content, secretome, metabolic pathways and annotation of eukaryote protein kinases (ePKs). S.F.C.,
559 L.Y., D.L., M.Z. and D.McM conducted pheroreception studies. M.T.S., K.K.G., U.N. and K.F.H 560 conducted bacterial symbiont analysis. S.L., S-M.Z., E.S.L. and B.C.B. performed virus analyses.
561 M.K., P.F., W.I. and N.R. performed annotation of HSP. T.P.Y., X-J.W., U.B-W. and N.D.
562 conducted proteogenomic studies of parasite-reactive snail host proteins and data analysis. R.F., A.
563 E.L. and C.S.J. performed annotation of CYP. J.H. performed annotation of NFkB. K.M.B. and
564 J.P.R. performed annotation of conserved immune factors. C.M.A. and J.J.P performed annotation of
565 FREPs. M.C. and C.M. performed annotation of complement. D.D. performed annotation of
566 apoptosis. B.G. and C.J.B. performed annotation of REDOX balance. O.L.B., D.D., R.G., Ch.C. and
567 G.M. performed annotation of antibacterial defenses. L.d.S. and A.T.P performed search for
568 antibacterial defense genes. P.C.H., M.A.G. and E.A.P. performed annotation of unknown novel
569 sequences. K.K.G., I.W.C., U.N., K.F.H., Ce.C., T.Q. and C.G. performed annotation and analysis of
570 epigenetic sequences. E.H.B., L.R. doA., M.deS.G., R.L.C, and W.deJ.J. performed annotation of
571 miRNA (Brazil), K.K.B., R.P. and K.B.S. performed annotation of miRNA (Canada). M.G.
572 performed annotation of periodicity. S.F.C., B.R., T.W., A.E.L and S.K.conducted neuropeptide
573 studies and data analysis. A.E.L., R.F., S.K., E.J.R., S.J., D.R., C.S.J. and L.R.N.performed
574 annotation neuroendocrinology (CYP). J.M.K., B.R. and S.F.C.performed annotation of ovipostatin.
575 A.B.K and L.L.M performed tissue location of transcripts analysis. A.J.W. and S.P.L. performed
576 annotation of phosphatases. T.L.L., K.M.R., M.Mi., and R.C. performed annotation of actins and
577 annotation of cardiac transcriptional program with D.L.T. D.J.J., B.K. and M.Me. performed
578 annotation of biomineralization genes. J.C., A.K. and C.F. performed annotation of DNA
579 transposons, global analysis of transposable element landscape, and horizontal transfer events. A.S
580 and C.B. performed repeat/TE analysis. E.S.L, S.M.Z., G.M.M and SKB conducted comparative B.
581 pfeifferi transcriptome studies and data analysis. C.M.A, P.M., M.L.M did most of the writing with
582 contributions from all authors.
583 584 Author Information
585 The Biomphalaria glabrata genome project has been deposited at DDBJ/EMBL/
586 GenBank under the accession number APKA00000000.1. All short-read data have been
587 deposited into the Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra)
588 as follows: Illumina HiSeq 2000 WGS reads:SRX648260-71; 454 GS FLX WGS: SRX005828,
589 SRX008161,2; 454 GS FLX RNA reads: SRX014813, SRX014894-7
590 Genome, transcriptome and predicted proteome data are also available at Vectorbase (ref. 47).
591 Reprints and permissions information is available at www.nature.com/reprints.
592 The authors declare no competing financial interests. Readers are welcome to comment on the online
593 version of the paper. Correspondence and requests for materials should be addressed to C.M.A.
594 ([email protected]).
595 596 597 List of Supplementary Files 598
599 PDF file 600 601 1. Supplementary information (SIZE) 602 This file contains Supplementary Text and Data sections 1-32 (see Contents list for details), 603 Supplementary Figures 1-87 604
605 Zip files 606 1. Supplementary Figure 8 (JPG, 8500 KB). 607 608 2 Supplementary Tables 1-56 (Excel 1919048 KB).